Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/03/13

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(03/13/13)
Current revision (23:31, 12 March 2013) (view source)
(03/13/13)
 

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03/13/13

  • Golden Gate assembly



Golden Gate Assembly

  • First, make fresh 10x Promega Buffer:
Reagent Conc. Volume
1M Tris-HCl (pH 7.8) 300 mM 300.0 μL
1M MgCl2 100 mM 100.0
1M DTT 100 mM 100.0
ATP 10 mM 10.0
dH2O 490.0
Final vol. 1000 uL


  • Golden Gate assembly reactions
  1. Promega - H2B + LOV-his + pSB1A3
  2. Promega - pSB1A3
  3. Roche - H2B + LOV-his + pSB1A3
  4. Roche - pSB1A3


Reagent Promega (1) Promega (2) Roche (9, 10) Roche (11, 12)
gg2 pSB1A3* 1.0 1.0 1.0 1.0
gg3 hPCD* 1.0 --- 1.0 ---
gg4 BL01* 1.0 --- 1.0 ---
ligase buffer 1.0 1.0 5.0 5.0
NEB T4 ligase 0.25 0.25 0.25 0.25
NEB BsmBI 0.5 0.5 0.5 0.5
dH2O 5.25 7.25 1.25 3.25
  10.0 10.0 10.0 10.0

Note: *DNA is 20 fmole/μL

Thermal cycler

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.
  • 4°C, ∞


  • Transformations
    • 50 μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar




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